Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy

The diffusion of macromolecules in cells and in complex fluids is often found to deviate from simple Fickian diffusion. One explanation offered for this behavior is that molecular crowding renders diffusion anomalous, where the mean-squared displacement of the particles scales as $\langle r^2 \rangle \propto t^{\alpha}$ with $\alpha < 1$. Unfortunately, methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) probe diffusion only over a narrow range of lengthscales and cannot directly test the dependence of the mean-squared displacement (MSD) on time. Here we show that variable-lengthscale FCS (VLS-FCS), where the volume of observation is varied over several orders of magnitude, combined with a numerical inversion procedure of the correlation data, allows retrieving the MSD for up to five decades in time, bridging the gap between diffusion experiments performed at different lengthscales. In addition, we show that VLS-FCS provides a way to assess whether the propagator associated with the diffusion is Gaussian or non-Gaussian. We used VLS-FCS to investigate two systems where anomalous diffusion had been previously reported. In the case of dense cross-linked agarose gels, the measured MSD confirmed that the diffusion of small beads was anomalous at short lengthscales, with a cross-over to simple diffusion around $\approx 1~\mu$m, consistent with a caged diffusion process. On the other hand, for solutions crowded with marginally entangled dextran molecules, we uncovered an apparent discrepancy between the MSD, found to be linear, and the propagators at short lengthscales, found to be non-Gaussian. These contradicting features call to mind the "anomalous, yet Brownian" diffusion observed in several biological systems, and the recently proposed "diffusing diffusivity" model

An ultrasonic orthopaedic surgical device based on a cymbal transducer

AbstractAn ultrasonic orthopaedic surgical device is presented, where the ultrasonic actuation relies on a modification of the classical cymbal transducer. All current devices consist of a Langevin ultrasonic transducer with a tuned cutting blade attached, where resonance is required to provide sufficient vibrational amplitude to cut bone. However, this requirement restricts the geometry and offers little opportunity to propose miniaturised devices or complex blades. The class V flextensional cymbal transducer is proposed here as the basis for a new design, where the cymbal delivers the required vibrational amplitude, and the design of the attached cutting insert can be tailored for the required cut. Consequently, the device can be optimised to deliver an accurate and precise cutting capability. A prototype device is presented, based on the cymbal configuration and designed to operate at 25.5kHz with a displacement amplitude of 30μm at 300V. Measurements of vibrational and impedance responses elucidate the mechanical and electrical characteristics of the device. Subsequent cutting tests on rat femur demonstrate device performance consistent with a commercial Langevin-based ultrasonic device and show that cutting is achieved using less electrical power and a lower piezoceramic volume. Histological analysis exhibits a higher proportion of live cells in the region around the cut site for the cymbal device than for a powered sagittal or a manual saw, demonstrating the potential for the ultrasonic device to result in faster healing

Telomerase Inhibition Targets Clonogenic Multiple Myeloma Cells through Telomere Length-Dependent and Independent Mechanisms

Plasma cells constitute the majority of tumor cells in multiple myeloma (MM) but lack the potential for sustained clonogenic growth. In contrast, clonotypic B cells can engraft and recapitulate disease in immunodeficient mice suggesting they serve as the MM cancer stem cell (CSC). These tumor initiating B cells also share functional features with normal stem cells such as drug resistance and self-renewal potential. Therefore, the cellular processes that regulate normal stem cells may serve as therapeutic targets in MM. Telomerase activity is required for the maintenance of normal adult stem cells, and we examined the activity of the telomerase inhibitor imetelstat against MM CSC. Moreover, we carried out both long and short-term inhibition studies to examine telomere length-dependent and independent activities.Human MM CSC were isolated from cell lines and primary clinical specimens and treated with imetelstat, a specific inhibitor of the reverse transcriptase activity of telomerase. Two weeks of exposure to imetelstat resulted in a significant reduction in telomere length and the inhibition of clonogenic MM growth both in vitro and in vivo. In addition to these relatively long-term effects, 72 hours of imetelstat treatment inhibited clonogenic growth that was associated with MM CSC differentiation based on expression of the plasma cell antigen CD138 and the stem cell marker aldehyde dehydrogenase. Short-term treatment of MM CSC also decreased the expression of genes typically expressed by stem cells (OCT3/4, SOX2, NANOG, and BMI1) as revealed by quantitative real-time PCR.Telomerase activity regulates the clonogenic growth of MM CSC. Moreover, reductions in MM growth following both long and short-term telomerase inhibition suggest that it impacts CSC through telomere length-dependent and independent mechanisms

In Vivo Inhibition Of Lung Cancer By Grn163L: A Novel Human Telomerase Inhibitor

Differential regulation of telomerase activity in normal and tumor cells provides a rationale for the design of new classes of telomerase inhibitors. The telomerase enzyme complex presents multiple potential sites for the development of inhibitors. GRN163L, a telomerase enzyme antagonist, is a lipid-modified 13-mer oligonucleotide N3' -> P5'-thiophosphoramidate, complementary to the template region of telomerase RNA (hTR). We evaluated both the in vitro and in vivo effects of GRN163L using A549-luciferase (A549-Luc) human lung cancer cells expressing a luciferase reporter. GRN163L (1 mu mol/L) effectively inhibits telomerase activity of A549-Luc cells, resulting in progressive telomere shortening. GRN163L treatment also reduces colony formation in soft agar assays. Surprisingly, after only I week of treatment with GRN163L, A549-Luc cells were unable to form robust colonies in the clonal. efficiency assay, whereas the mismatch control compound had no effect. Finally, we show that in vivo treatment with GRN163L is effective in preventing lung metastases in xenograft animal models. These in vitro and in vivo data support the development of GRN163L as a therapeutic for the treatment of cancer.WoSScopu

Telomerase is active and can be inhibited in MM CD138⁺ and CD138^neg cells.

<p>(A) Relative telomerase activity in CD138⁺ plasma cells and CD138^neg CSC isolated from MM cell lines. (B) Relative telomerase activity in CD138⁺ plasma cells and CD19⁺CD27⁺ CSC isolated from clinical MM bone marrow samples. (C) Relative telomerase activity in bulk MM cell lines following 72 hours of treatment with vehicle, mismatch control oligonucleotide, or imetelstat (1 uM). (D) Relative telomerase activity in CD138⁺ and CD138^neg NCI-H929 cells after 72 hours of treatment with mismatch control oligonucleotide or imetelstat (1 uM).</p

Prolonged telomerase inhibition decreases MM CSC telomere length and clonogenic potential.

<p>(A) Percentage of telomeres less than 1.4 kb as determined by STELA after 1, 2, or 3 weeks with mismatch control oligonucleotide or imetelstat (*p-value <0.05 and NS; not significant). (B) Relative clonogenic growth of CD138^neg NCI-H929 and RPMI8226 cells after 3 and 5 weeks of treatment with mismatch control oligonucleotide or imetelstat (1 uM). (C) Relative clonogenic recovery of CD138^neg cells isolated from primary clinical specimens following 3 weeks of treatment with vehicle, mismatch control oligonucleotide or imetelstat (1 uM). (D) Survival of NOD/SCID mice injected with NCI-H929 cells then treated with imetelstat (solid line) or mismatch control oligonucleotide (dashed line) for two weeks <i>in vivo</i> (p<0.01, n = 8,). (E) Survival of NOD/SCID mice after injection with NCI-H929 cells following in vitro treatment with imetelstat (solid line) or mismatch control oligonucleotide (dashed line) for two weeks (<i>P</i><0.001, n = 20 per group).</p

Spatiotemporal Modeling of Zoonotic Arbovirus Transmission in Northeastern Florida Using Sentinel Chicken Surveillance and Earth Observation Data

The irregular timing and spatial variation in the zoonotic arbovirus spillover from vertebrate hosts to humans and livestock present challenges to predicting spillover occurrence over time and across broader geographic areas, compromising effective prevention and control strategies. The objective of this study was to quantify the effects of the landscape composition and configuration and dynamic weather events on the 2018 spatiotemporal distribution of eastern equine encephalitis virus (EEEV) (Togaviridae, Alphavirus) and West Nile virus (WNV) (Flaviviridae, Flavivirus) sentinel chicken seroconversion in northeastern Florida. We used a modeling framework that explicitly accounts for joint spatial and temporal effects and incorporates key EO (Earth Observation) information on the climate and landscape in order to more accurately quantify the environmental effects on the transmission to sentinel chickens. We investigated the environmental effects using Bernoulli generalized linear mixed effects models (GLMMs), including a site-level random effect, and then added spatial random effects and spatiotemporal random effects in subsequent runs. The models were executed using an integrated nested Laplace approximation (INLA) and a stochastic partial differential equation (SPDE) approach in R-INLA. The GLMMs that included a spatiotemporal random effect performed better relative to models that included only spatial random effects and also performed better than non-spatial models. The results indicated a strong spatiotemporal structure in the seroconversion for both viruses, but EEEV exhibited a more punctuated and compact structure at the beginning of the sampling season, while WNV exhibited a more gradual and diffuse structure across the study area toward the end of the sampling season. The percentage of cypress&ndash;tupelo wetland land cover within 3500 m of coop sites and the edge density of the forest land cover within 500 m had a strong positive effect on the EEEV seroconversion, while the best fitting model for WNV was the intercept-only model with spatiotemporal random effects. The lagged climatic variables included in our study did not have a strong effect on the seroconversion for either virus when accounting for temporal autocorrelation, demonstrating the utility of capturing this structure to avoid type I errors. The predictive accuracy for out-of-sample data for the EEEV seroconversion demonstrates the potential to develop a framework that incorporates temporal dynamics in order to better predict arbovirus transmission